The protozoan parasite Toxoplasma gondii is an important opportunistic parasite causing life threatening infections in the immune compromised and severe birth defects when acquired in utero. The parasite grows within a specialized compartment, the parasitophorous vacuole (PV) within the infected host cell. The boundary defining this niche is the PV membrane (PVM) that serves as the interface between the parasite and host. Despite its importance, investigation of the PVM has been limited. We have recently completed a proteomic analysis of the T. gondii PVM resulting in the identification of 123 proteins of which 70 appear to be novel. Of these novel proteins, 26 are annotated as hypothetical ORF's precluding any assignment of function. As hypothetical ORF's, these loci lack any identifiable motif indicating they are truly novel. While several appear unique to Toxoplasma, others have similarly annotated homologs in other less tractable Apicomplexa including Plasmodium and Cryptosporidium spp. suggesting conserved function. As novel proteins that are potentially localized to the PVM, an organelle found only in the context of parasite infection, these hypothetical ORF's represent potentially unique activities that may serve as drug targets. In this study we exploit recently developed technologies to epitope tag and localize the hypothetical ORF's as a prelude to cloning the validated genes and attempting the gene knockouts. Together these studies represent a secondary analysis of an existing data set as a limited short term project with a high potential of revealing novel activities. As such it is ideally suited to the RO3 funding mechanism. With this proposal we aim to perform the initial characterization of these orphan genes toward establishing their functions in parasite biology. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii causes serious illness in immune compromised individuals and is a model organism for several less experimentally tractable parasites. This study aims to investigate truly novel proteins that were identified as being components of the parasitophorous vacuole membrane, a unique and essential parasite organelle. We expect with the completion of this work to open new areas of investigation in parasite biology.